Hyaluronic acid gel, a method of its production and medical material containing it

ABSTRACT

A gel made of hyaluronic acid alone which is hardly soluble in a neutral aqueous solution and which keeps its shape for at least one day in a neutral aqueous solution at 25° C.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates to a novel hyaluronic acid gel and amethod of its production, and further, to a biomedical material withgood biocompatibility.

2. Background Art

Hyaluronic acid is a linear macromolecular polysaccharide consisting ofalternately bonded β-D-N-acetylglucoamine and β-D-glucuronic acid.Hyaluronic acid is found not only in connective tissues of mammals butalso in cockscombs and the capsules of streptococci. Hyaluronic acid isobtainable not only by extraction from cockscombs and umbilical cords,but also as purified products from the culture broth of streptococci.

Natural hyaluronic acid is polydisperse in respect of molecular weightand is known to show excellent biocompatibility even when implanted orinjected into the body by virtue of the absence of species and organspecificity. Further, because of the drawbacks of hyaluronic acid inbiological application attributable to the easiness of dissolution inwater such as the relatively short in vivo residence time, variouschemical modifications of hyaluronic acid have been proposed.

A representative of them is a high-swelling crosslinked hyaluronic acidgel obtained by using a bifunctional crosslinker such as divinylsulfone, a bisepoxide or formaldehyde (U.S. Pat. No. 4,582,865,JP-B-6-37575, JP-A-7-97401 and JP-A-60-130601).

A chemical modification of hyaluronic acid utilizing the solubility oftetrabutylammonium hyaluronate in organic solvents such as dimethylsulfoxide has been disclosed, too (JP-A-3-105003). Formation of esterlinkages between the carboxyl groups and the hydroxyl groups inhyaluronic acid by treating tetrabutylammonium hyaluronate withtriethylamine and 2-chloro-1-methylpyridinium iodide in dimethylsulfoxide has also been disclosed (EP-A-0341745A1).

Further, as an approach to insolubilization of hyaluronic acid in waterwithout using covalently binding chemicals, preparation of a hyaluronicacid-polymer complex by ionically bonding hyaluronic acid and a polymerhaving an amino or imino group via the carboxyl groups in hyaluronicacid and the amino or imino group in the polymer has been disclosed(JP-A-6-73103).

It is known that a hyaluronic acid aqueous solution forms a so-calledputty gel by jellying when acidified, for example, to pH 2.0-2.7, but noputty gel is formed at a pH below 2.

The putty gel is differentiated from the hyaluronic acid gel accordingto the present invention by its quick dissolution in a neutral aqueoussolution.

As another approach, production of a hyaluronic acid gel from ahyaluronic acid aqueous solution in the presence of 20-80 wt % of awater-miscible organic solvent at pH 2.0-3.8 has been disclosed(JP-A-5-58881). However, it is also disclosed that the resultinghyaluronic acid gel dissolves in water with no coating on it.

Further, some general methods of producing polymer gels by repeatedlyfreezing and thawing aqueous solutions of polymers represented bypolyvinyl alcohol and glucomannan have been proposed (JP-A-57-190072 andJP-A-5-161459).

Although freezing-thawing and freeze-drying are widely used as generaltechniques for purifying or preserving hyaluronic acid or biogenicsamples containing hyaluronic acid, no report has been made on formationof a hyaluronic acid gel by such techniques yet because they are usuallyused under neutrality control.

Hyaluronic acid has extraordinarily high viscosity and good moistureretentivity, and is intrinsically devoid of antigenicity and highlybiocompatible. Therefore, it is used as a therapeutic medicine forosteoarthritis and as a supplementary material in ophthalmic surgery.

Use of hyaluronic acid itself as a postoperative adhesion preventive hasalso been studied. However, hyaluronic acid does not have much effectdue to the relatively short in vivo residence time and diffusivelydrains away from the wound surface in a short time due to its watersolubility (Journal of Gynecologic Surgery vol.7, No.2, 97-101(1991)).

Modification of carboxymethyl cellulose and sodium hyaluronate with acarbodiimide crosslinker on the basis of JP-A-5-508161 and JP-A-6-508169afforded the development of an adhesion preventive film “Seprafilm”(Genzyme).

Despite attempts to utilize the outstanding biocompatibility intrinsicto hyaluronic acid to the maximum, no hyaluronic acid gel usable as abiocompatible biomedical material with a long in vivo residence time hasbeen developed yet without any chemical crosslinkers or chemicalmodifiers or formation of complexes with cationic polymers.

The present inventors have conducted extensive research on thephysicochemical properties of hyaluronic acid itself and consequentlyhave found that a hyaluronic acid gel can be obtained by freezing andthawing at least once a hyaluronic acid aqueous solution adjusted to aspecific pH. They have also found that the hyaluronic acid gel thusobtained dissolves in water very slowly.

Conventional modifications of hyaluronic acid have an inevitable problemof extra risks such as toxicity and bioincompatibility intrinsic to themodifications because of the use of chemical reactants despitenumberless efforts.

For example, chemical modification, crosslinking or ionic treatment ofhyaluronic acid with a metal salt may afford adhesion preventives withimproved in vivo persistency. However, the resulting adhesionpreventives no longer retain the structure of natural hyaluronic acidand are not essentially the same as natural hyaluronic acid in respectof physiological effects, biocompatibility and safety inclusive oftoxicity, because of the crosslinkers or metals covalently or ionicallybound in the hyaluronic acid molecules. In addition, it has beendifficult to completely circumvent the problems of the residual toxicityof these crosslinkers and the risk of decomposition products ofcrosslinkers to the body.

DISCLOSURE OF THE INVENTION

The present inventors have found that the hyaluronic acid gel accordingto the present invention has ideal biocompatibility and persistency as abiomedical material, particularly ideal biocompatibility and persistencyas an adhesion preventive and markedly prevents postoperative adhesion.The present inventors have accomplished the present invention on thebasis of this discovery.

The present invention provides (1) a gel made of hyaluronic acid alonewhich is hardly soluble in a neutral aqueous solution, (2) thehyaluronic acid gel according to (1), which keeps its shape for at leastone day in a neutral aqueous solution at 25° C., (3) the hyaluronic acidgel according to (1), which dissolves in a neutral aqueous solution at25° C. in one day to a degree of dissolution of at most 50%, (4) thehyaluronic acid gel according to (1), which dissolves in a neutralaqueous solution at 37° C. in 12 hours to a degree of dissolution of atmost 50%, (5) the hyaluronic acid gel according to (1), which dissolvesto yield solubilized hyaluronic acid having a branched structure andpartly containing a molecular weight fraction with a branching degree ofat least 0.5, when treated under accelerating conditions for acidhydrolysis of hyaluronic acid, (6) the hyaluronic acid gel according to(1), which is formed by freezing and then thawing an aqueous solution ofhyaluronic acid at pH 3.5 or below, (7) a method of producing thehyaluronic acid gel according to (6), which comprises adjusting anaqueous solution of hyaluronic acid to pH 3.5 or below, and freezing andthawing the solution at least once, (8) a biomedical material containinga gel made of hyaluronic acid alone which satisfies the followingrequirements (a) and (b): (a) the hyaluronic acid gel dissolves in aneutral aqueous solution at 25° C. in one day to a degree of dissolutionof at most 50%, and (b) the gel dissolves to yield solubilizedhyaluronic acid having a branched structure and partly containing amolecular weight fraction with a branching degree of at least 0.5, whentreated under accelerating conditions for acid hydrolysis of hyaluronicacid, (9) the biomedical material according to (8), wherein the gel madeof hyaluronic acid alone is sheet-like, filmy, flaky, spongy, massive,fibrous or tubular, (10) a biomedical material containing a hyaluronicacid gel and un-gelled hyaluronic acid, wherein the hyaluronic acid geldissolves in a neutral aqueous solution at most 50%, and the hyaluronicacid gel dissolves to yield solubilized hyaluronic acid having abranched structure and partly containing a molecular weight fractionwith a branching degree of at least 0.5, when treated under acceleratingconditions for acid hydrolysis of hyaluronic acid, (11) a biomedicalmaterial containing a hyaluronic acid gel made of hyaluronic acid alonewhich is sheet-like, filmy, spongy, massive, fibrous or tubular andun-gelled hyaluronic acid, and (12) the biomedical material according toany one of (8) to (11), which is an adhesion preventive.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph that shows the comparison between GPC chromatogramsand the molecular weights of the respective fractions obtained inExample 8 and Comparative Example 6.

FIG. 2 is a graph that shows the relation between the branching degreeand the molecular weight in Example 8 on the basis that the hyaluronicacid in Comparative Example 6 was linear.

BEST MODE FOR CARRYING OUT THE INVENTION

Now, the present invention will be described below in detail.

In the present invention, hyaluronic acid obtained by extraction fromanimal tissues or by fermentation may be used without any restriction onits source.

The strain used in fermentation is preferably a hyaluronicacid-producing microorganism isolated from nature such as the genusStreptococcus or a mutant which steadily produces hyaluronic acid inhigh yield such as Streptococcus equi FM-100 (accession number 9027given by National Institute of Bioscience and Human-Technology)disclosed in JP-A-63-123392 or Streptococcus equi FM-300 (accessionnumber 2319 given by National Institute of Bioscience andHuman-Technology) disclosed in JP-A-2-234689. Pure hyaluronic acidobtained from cultures of the above-mentioned mutants may be used.

Gel is defined as “a polymer having a three-dimensional networkstructure insoluble in any solvent or its swollen product” byEncyclopedia of Polymer (Kobunshi Jiten) New Edition (published byAsakura Shoten, 1988). It is also defined as “a jellied product of a sol(a colloidal solution)” by Encyclopedia of Science and Chemistry(Rikagaku Jiten) Forth Edition (published by Iwanami Shoten, 1987).

The hyaluronic acid gel according to the present invention ischaracterized in that it is hardly soluble in a neutral aqueoussolution, and when the hyaluronic acid gel is put in a neutral aqueoussolution, it dissolves with significantly greater difficulty thanun-gelled hyaluronic acid does. The difficulty in dissolution is definedby the persistence of the shape of the gel and the solubility of the gelin a neutral aqueous solution at 25° C. and the solubility of the gel ina neutral aqueous solution at 37° C. The neutral aqueous solution meansa buffered physiological saline adjusted to pH 7.

The hyaluronic acid gel according to the present invention is alsocharacterized in that it dissolves quickly in an aqueous alkaline buffersolution, for example, at pH 11.

The hyaluronic acid gel according to the present invention is a polymerhaving a three dimensional network structure or its swollen product. Thethree dimensional network structure is made of crosslinked hyaluronicacid.

The hyaluronic acid gel according to the present invention can besolubilized through degradation by treatment under acceleratingconditions for acid hydrolysis of hyaluronic acid. When the solubilizedhyaluronic acid retains the crosslinked structure, it is distinguishedas branched hyaluronic acid from linear hyaluronic acid according to thetheory of polymer solution. Hyaluronic acid itself is a linear polymerand known to have no branches (Biochemistry of Polysaccharides IChemistry Version (Tato Seikagaku I Kagaku-hen) published by KyoritusShuppan, 1969).

The accelerating conditions for acid hydrolysis of hyaluronic acidaccording to the present invention are preferably such that the pH ofthe aqueous solution is 1.5 and the temperature is 60° C. It is wellknown that cleavage of the main chain of hyaluronic acid throughhydrolysis of glycosidic bonds is remarkably accelerated in an acidic oralkaline aqueous solution as compared with that in a neutral aqueoussolution (Eur. Polym. J. Vol32, No8, p1011-1014, 1996). In addition,acid hydrolysis is accelerated at a higher temperature.

The reaction time for the accelerated hydrolysis of a hyaluronic acidgel according to the present invention heavily depends on the structureof the hyaluronic acid gel such as the molecular weight or molecularweight distribution of the hyaluronic acid as the raw material, anddegree of crosslinking of the gel.

The reaction conditions are selected so that the proportion of thesolubilized hyaluronic acid and the branching degree are large. If thereaction conditions are too mild or too severe, the branching degree isdifficult to measure, because the proportion of the solubilizedhyaluronic acid is small under mild conditions, and on the other hand,the molecular weight of the solubilized hyaluronic acid is too smallunder severe reaction conditions. Besides, the possibility ofdestruction of the branch points themselves increases.

As to the reaction conditions, a preferable reaction time is such thatthe visually recognizable hyaluronic acid gel disappears almostcompletely or such that the proportion of the solubilized hyaluronicacid reaches 50% or above.

For the measurement of the molecular weight and branching degree of thesolubilized hyaluronic acid, the GPC-MALLS method, which uses adifferential refractometer and a multi-angle laser light scatteringdetector (MALLS) as detectors for gel permeation chromatogram (GPC), theGPC-LALLS method, which uses a differential refractometer and a lowangle laser light scattering detector as detectors for GPC, and theGPC-viscosity method, which uses a differential refractometer and aviscometer as detectors for GPC, may be mentioned.

In the present invention, the molecular weights and branching degrees ofthe fractions separated by GPC according to molecular weight aremeasured on-line continuously by the GPC-MALLS method. The GPC-MALLSmethod allows continuous measurement of the molecular weight and radiusof gyration of each fraction separated by GPC. In the GPC-MALLS method,for calculation of the branching degree, two methods are available: theradius of gyration method which compares the correlation between themolecular weight and radius of gyration of the fractionated solubilizedhyaluronic acid with the correlation of the molecular weight and radiusof gyration of fractionated linear hyaluronic acid as a control, and theelution volume method which compares the molecular weight of eachfraction of the solubilized hyaluronic acid with the molecular weight ofa fraction at the same elution volume of linear hyaluronic acid as acontrol.

In the present invention, the branching degree was measured by theelution volume method. The branching degree is the number of branchpoints in one polymer chain of the solubilized hyaluronic acid andplotted against the molecular weight of the solubilized hyaluronic acid.

For determination of the molecular weight and radius of gyration of eachfraction, the Zimm plot of the equation (1) at finite concentration wasused. The molecular weight was calculated from the extrapolation toscattering angle 0°, and the radium of gyration was calculated from theangle-dependent initial slope in accordance with the followingequations. $\begin{matrix}\begin{matrix}\begin{matrix}{\frac{K_{c}}{R(\theta)} = {\frac{1}{{MP}(\theta)} + {2A_{2}c} + \ldots}} \\{{P(\theta)}^{- 1} = {1 + {{{1/3} \cdot k^{2}}{\langle S^{2}\rangle}} + \ldots}}\end{matrix} \\{k = {\frac{4\pi}{\lambda}{\sin \left( {\theta/2} \right)}}}\end{matrix} & (1)\end{matrix}$

wherein M is the molecular weight, <S²> is the mean square radius ofgyration, K is an optical constant, R(θ) is the reduced excessscattering intensity, c is the polymer concentration, P(θ) is theparticle scattering function, λ is the wavelength of the laser beam inthe solution, and A₂ is the second virial coefficient, 0.002 ml·mol/g²for hyaluronic acid. c is calculated from the output of a differentialrefractometer, based on the differential refractive index increment of ahyaluronic acid aqueous solution (dn/dc: 0.153 ml/g).

In the GPC-MALLS method, the molecular weight and mean square radius ofgyration are calculated from the reduced excess scattering intensity,and therefore, the measuring accuracy depends on the reduced excessscattering intensity. Equation (1) relates the reduced excess scatteringintensity to both the concentration and the molecular weight.Accordingly, the sample concentration and the injection volume must bedetermined in accordance with the molecular weight of the sample. Whenthe GPC column for molecular weight fractionation is selected, themaximum sample concentration and injection volume should be selected sothat the GPC column is not overloaded.

In the elution volume method, the branching degree of each fraction wascalculated in accordance with equation (2) given below. The shrinkagefactor, g, is determinable from the molecular weights of a branchedpolymer, M_(b), and a linear polymer, M₁, in fractions at the sameelution volume.

g=(M ₁ /M _(b))^((a+1)/e)  (2)

Here, a is the Mark-Houwink constant, which is 0.78 for hyaluronic acid,and e is the draining factor, which is defined as 1.

When randomly branched polymer (long chain branching, tetrafunctional)are assumed, the number of branches in one polymer chain, B, (branchingdegree) can be calculated in accordance with equation (3) given below.$\begin{matrix}{g = \frac{1}{\left\lbrack {\left\lbrack {1 + \frac{B}{6}} \right\rbrack^{0.5}\left\lbrack \frac{4B}{3\pi} \right\rbrack} \right\rbrack^{0.5}}} & (3)\end{matrix}$

Measurement of branching degree by the elution volume method is the sameas measurement of branching degree by the GPC-LALLS method, details ofwhich are found in Size Exclusion Chromatography (Kyoritsu Shuppan,1991).

Solubilized hyaluronic acid was diluted with the GPC eluent forconcentration adjustment and filtered through a membrane filter of 0.2μm before measurement.

If the hyaluronic acid gel according to the present invention has acrosslinked structure which is stable under accelerating conditions foracid hydrolysis of hyaluronic acid, a branched structure is recognizedin the solubilized hyaluronic acid according to the theory of polymersolution.

In the present invention, by hyaluronic acid alone, it is meant that nochemical crosslinker or chemical modifier is used other than hyaluronicacid, and that hyaluronic acid is not in the form of a complex with acationic polymer.

The chemical crosslinker for hyaluronic acid means a polyvalent compoundwhich reacts with the carboxylic group, hydroxyl group or acetamidogroup in hyaluronic acid to form a covalent bond. For example, apolyvalent epoxy compound such as polyglycidyl ether, divinyl sulfone,formaldehyde, phosphorus oxychloride, the combination of a carbodiimidecompound and an amino acid ester, and the combination of a carbodiimidecompound and a dihydrazide compound may be mentioned. A chemicalcrosslinker reacts with hyaluronic acid to form a three-dimensionalnetwork structure.

The chemical modifier for hyaluronic acid means a compound which reactswith the carboxylic group, hydroxyl group or acetamido group inhyaluronic acid to form a covalent bond. For example, the combination ofacetic anhydride and concentrated sulfuric acid, the combination oftrifluoroacetic anhydride and an organic acid and an alkyl iodidecompound may be mentioned. It makes the hydrophilic groups in hyaluronicacid hydrophobic and thereby lowers the solubility of hyaluronic acid.

The cationic polymer which forms a complex with hyaluronic acid means apolymer which forms a complex through an ionic bond between thecarboxylic groups in hyaluronic acid and the amino or imino group in thepolymer, and chitosan, polylysine, polyvinylpyridine, polyethyleneimineand polydimethylaminoethylmethacrylate may be mentioned, for example. Acationic polymer and hyaluronic acid form a complex insoluble in water.

On the other hand, substances which do not directly induce introductionof a crosslinked structure into hyaluronic acid or make hyaluronic acidinsoluble or hardly soluble may be added when the hyaluronic acid gelaccording to the present invention is prepared. Materials asbiocompatible as hyaluronic acid such as chondroitin sulfate andcarboxymethyl cellulose, may be mixed or incorporated to give ahyaluronic acid gel without any restriction.

Further, in preparation of a hyaluronic acid gel, pharmacologically orphysiologically active substances may be added to give a hyaluronic acidgel containing such substances without any restriction.

The molecular weight of the hyaluronic acid to be used in the presentinvention is preferably within the range of from about 1×10⁵ to about1×10⁷ Da. Hyaluronic acid having a higher molecular weight may also beused after the molecular weight is lowered into this range by treatmentsuch as hydrolysis.

In the present invention, the concept of hyaluronic acid is used so asto include its alkali metal salts such as sodium, potassium and lithiumsalts, too.

The hyaluronic acid aqueous solution used in the present invention isobtained by stirring a mixture of powdery hyaluronic acid and water. Thehyaluronic acid concentration is preferably 5.0 wt % or less in view ofhandling of the aqueous solution.

When hyaluronic acid having a molecular weight of 2×10⁶ Da or more isused, the concentration is preferably 2.5 wt % or less.

For pH adjustment of a hyaluronic acid aqueous solution, any acid thatcan adjust the pH to 3.5 or below may be used. Preferably, a strong acidsuch as hydrochloric acid, nitric acid and sulfuric acid is used todecrease the amount of an acid.

The pH of a hyaluronic acid aqueous solution is adjusted so that asufficient proportion of the carboxylic groups in hyaluronic acidundergoes protonation. The dissociation constant of hyaluronic acid inthe acid form is logK₀=4.25 when hyaluronic acid is diluted toindefinite concentrations (Acta Chemica Hungarica-Models in Chemistry129(5), 671-683, 1992). In the present invention, it is necessary toadjust the pH to 3.5 or below, preferably to 2.5 or below, although thefinal pH is set depending on the type of the counterinon in thehyaluronic acid salt, the molecular weight of hyaluronic acid, theconcentration of the aqueous solution, conditions of freezing andthawing, and the properties of the resulting gel such as strength.

With respect to freezing-thawing, a procedure comprising freezing theprepared acidic hyaluronic acid aqueous solution in an appropriatevessel at a predetermined temperature and then thawing it at apredetermined temperature is carried out at least once. Although thefreezing and thawing temperatures and times may be appropriately setdepending on the size of the vessel and the volume of the aqueoussolution so that the acidic hyaluronic acid solution freezes and thaws,it is generally preferred that the freezing temperature is not higherthan the ice point, and the thawing temperature is not lower than theice point.

It is particularly preferred that the freezing temperature is −5° C. orbelow, and the thawing temperature is 5° C. or above, to shorten thefreezing and thawing times. There is no restriction on the freezing andthawing times so long as they are longer than it takes to completefreezing and thawing at the temperatures.

The number of times the procedure comprising freezing and then thawingthe prepared acidic hyaluronic acid aqueous solution is repeated,depends on the molecular weight of hyaluronic acid to be used, theconcentration and pH of the aqueous solution, the freezing and thawingtemperatures and times and various properties of the resulting gel suchas strength. Usually, it is preferred to repeat the procedure at leastonce.

Further, the freezing and thawing temperatures and times may be changedevery time the freezing-thawing is repeated.

From the hyaluronic acid gel obtained by freezing and thawing a preparedacidic hyaluronic acid aqueous solution, the acid component added forthe acidification has to be removed in order to prevent acid hydrolysisof hyaluronic acid. For removal of the acid component, the gel isusually washed with an aqueous solvent, for example, water,physiological saline or a phosphate buffer, preferably physiologicalsaline or a phosphate buffer. There is no restriction on the aqueoussolvent so long as it does not functionally impair the hyaluronic acidgel.

Although there is no particular restriction on the washing method, abatch method, a filtration method or a method in which a solvent ispassed through a loaded column is usually used. The washing conditions,inclusive of the volume of the washing solvent and the times of washing,may be selected considering the shape and the use of the hyaluronic acidgel so that the concentration of the component to remove can be loweredto the desired level or below.

The washed hyaluronic acid gel is used as a biomedical material in animmersed state with a solvent, in a wet state with a solvent or in a drystate after air-drying, vacuum drying or freeze drying depending on theuse.

With a view to shaping the hyaluronic acid gel, by selecting the vesseland the procedure when the prepared acidic hyaluronic acid aqueoussolution is frozen in its preparation, a hyaluronic acid gel of desiredshape such as a sheet-like, filmy, flaky, spongy, massive or tubularshape can be obtained. For example, freeze casting on a plate affords afilm or a sheet, and freezing-thawing in an organic solvent immisciblewith water under vigorous stirring affords flakes.

Preparation of a hyaluronic acid gel may be followed by post-treatmentsuch as mechanical fragmentation, rolling or spinning to make the gelinto fine flakes, a film or the like. However, a hyaluronic acid gel ofdesired shape can be obtained without any special treatment for shapingby appropriately selecting the production conditions. For example, aprepared acidic hyaluronic acid aqueous solution having a hyaluronicacid concentration of 0.1% or less, preferably 0.05% or below yields afine fibrous hyaluronic acid gel after freezing-thawing.

The hyaluronic acid gel obtained according to the present invention maybe used as a general biodegradable biomedical material in any fieldswherein hyaluronic acid is used without any particular restriction. Itmay be used for, for example, an adhesion preventive, a carrier for apharmacologically active substance, a wound dressing, an artificialskin, a replacement vital tissue repairer, a joint injection, a surgicalsuture, a hemostatic material, an artificial organ, an artificialextracellular matrix, an artificial basement membrane or biomedicalproducts such as medical tools and devices for diagnostic or therapeuticuse or medicinal compositions.

Shaped products of a hyaluronic acid gel may be used in combinations ofdifferent shapes let alone in a single shape and are expected to have astronger effect when mixed with or used in combination with an un-gelledhyaluronic acid.

For example, the combined use of a hyaluronic acid gel sheet and ahyaluronic acid solution as an adhesion preventive after peritoneotomyis expected to have both a regional effect and an extensiveintraperitoneal effect.

Also, a mixture of a flaky hyaluronic acid gel and a hyaluronic acidsolution is expected to have a rapid effect and a delayed effect as ajoint injection.

Now, the usefulness of the hyaluronic acid gel obtained according to thepresent invention as a biomedical material will be described withreference to the use as a slow release carrier for a pharmacologicallyactive substance.

The hyaluronic acid gel obtained according to the present invention canbe used as a carrier which slowly releases a pharmacologically activesubstance encapsulated in its structure. In this case, by controllingthe properties represented by degradability and shape of the hyaluronicacid gel in accordance with the kind of the pharmacologically activesubstance, the mode of use, the application site and the requiredresidence time, it is possible to adapt the gel to variouspharmacologically active substances and various modes of use.

When formulated appropriately, a pharmaceutical which can release apharmacologically active substance in a desired way can be obtained. Theslow release pharmaceutical may be administered orally, percutaneously,permucocutaneously, by injection or by implantation. Next, the adhesionpreventive of the present invention will be described below.

The adhesion preventive made of a hyaluronic acid gel obtained accordingto the present invention is a sheet-like, filmy, flaky, massive, fibrousor tubular material for surgical use. With respect to the mode of use,it is preferred to directly apply a filmy or sheet-like material to apart subjected to surgery. It is also preferred to apply a fine flakymaterial by injection to a part subjected to surgery. It is alsopreferably to peritoneoscopically apply a gel or a filmy material to apart subjected to surgery.

Further, an adhesion preventive made of a hyaluronic acid gelencapsulating a physiologically active substance can be obtained bymixing a prepared acidic hyaluronic acid solution and a physiologicallyactive substance and then freezing and thawing the mixture.

An adhesion preventive made of a hyaluronic acid gel is applicable toany animals that can suffer from adhesion and favorably preventspostoperative adhesion in mammals, especially in the human.

It is effective wherever it may be administered in the body, forexample, to various intaperitoneal and intrathoracic organs,peritenoneums, the skull, nerves and eyeballs in peritoneotomy,gynecological surgery and thoracotomy, to tendons and ligaments inorthopedic surgery and to the dura mater in neurosurgery.

The adhesion preventive made of a hyaluronic acid gel obtained accordingto the present invention may be administered at any time during or afteroperations so long as postoperative adhesion can be prevented, butpreferably at the last of an operation.

Now, the present invention will be described in further detail withreference to Examples. However, the present invention is by no meansrestricted to these specific Examples.

EXAMPLE 1

Sodium hyaluronate with a molecular weight of 2×10⁶ Da was dissolved indistilled water to give a 1 wt % hyaluronic acid aqueous solution. ThepH of the hyaluronic acid aqueous solution thus obtained was 6.0. The pHof the aqueous solution was adjusted to 1.5 with 1N hydrochloric acid. A15 ml portion of the acidic hyaluronic acid aqueous solution was put ina 30 ml glass bottle and placed in a refrigerator set at −20° C. for 16hours and then thawed at 25° C. to give a spongy hyaluronic acid gel.

EXAMPLE 2

In Example 1, the hyaluronic acid concentration was changed to 0.1 wt %in the preparation of the hyaluronic acid aqueous solution, and the sameprocedure as in Example 1 was followed to give a spongy hyaluronic acidgel.

EXAMPLE 3

In Example 1, hyaluronic acid with a molecular weight of 6×10⁵ Da wasdissolved to give a hyaluronic acid aqueous solution. After the sameadjustment operation as in Example 1, the aqueous solution thus obtainedwas placed in a refrigerator set at −20° C., and at least 6 hours offreezing and at least 2 hours of thawing at 25° C. were repeated 5 timesto give a spongy hyaluronic acid gel.

EXAMPLE 4

In Example 1, the freezing temperature was set at −10° C. Freezing at−10° C. for 77 hours and subsequent thawing at 25° C. gave a spongyhyaluronic acid gel.

EXAMPLE 5

In Example 1, an acidic hyaluronic acid aqueous solution at pH 2.5 wasprepared from a 0.4 wt % hyaluronic acid aqueous solution. A 15 mlportion of the acidic hyaluronic acid aqueous solution was put in a 30ml glass bottle and placed in a refrigerator set at −20° C. At least 6hours of freezing and at least 2 hours of thawing at 25° C. wererepeated 8 times to give a partially spongy hyaluronic acid gel.

COMPARATIVE EXAMPLE 1

In Example 1, a hyaluronic acid aqueous solution was frozen and thawedrepeatedly 8 times without pH adjustment. No change happened to thehyaluronic acid aqueous solution, namely gelation did not occur.

COMPARATIVE EXAMPLE 2

The hyaluronic acid aqueous solution prepared in Example 1 was air-driedat 60° C. to give a cast film of about 100 μm thick, which was subjectedto a solubility test for hyaluronic acid gels.

COMPARATIVE EXAMPLE 3

The hyaluronic acid aqueous solution prepared in Example 1 was frozen at−20° C. and freeze-dried to give a hyaluronic acid sponge, which wassubjected to a solubility test for hyaluronic acid gels.

EXAMPLE 6 Solubility Test for Hyaluronic Acid Gels

A phosphate buffer was added to physiological saline at a concentrationof 50 mM to give a phosphate buffer-physiological saline at pH 7.0. Thespongy hyaluronic acid gels obtained in the preceding Examples werewashed with distilled water and drained on filter paper. The hyaluronicacid gels were immersed in 50 ml of the phosphate buffer-physiologicalsaline based on 150 mg of dry hyaluronic acid in the gels.

The solids of hyaluronic acid obtained in Comparative Examples wereimmersed in 50 ml of the phosphate buffer-physiological saline based on150 mg of dry weight.

The degree of dissolution of hyaluronic acid in the phosphatebuffer-physiological saline at 25° C. was obtained from theconcentration of hyaluronic acid in the phosphate buffer-physiologicalsaline.

Namely, the solubility of a hyaluronic acid gel in a neutral aqueoussolution at 25° C. is defined according to this test.

Measurement of Hyaluronic Acid Concentration

The concentration of hyaluronic acid in the phosphatebuffer-physiological saline was obtained from the area of a GPC peak byusing a differential refractometer as a detector.

As described above, the solubility test was actually carried out on thehyaluronic acid gels obtained in Examples 1 to 4 and the solids ofhyaluronic acid obtained in Comparative Examples 2 and 3. The resultswere tabulated in Table 1 together with the results of observation ofthe shapes of the hyaluronic acid gels by the naked eye.

For example, in Test No.1, the degree of dissolution of the hyaluronicacid gel obtained in Example 1 was found to be 3% after 1 day, 5% after4 days and 6% after 7 days. Namely, 94% of the hyaluronic acid remainedeven after 7 days. The spongy shape was also maintained. In Test No.5,the degree of dissolution of the cast film of about 100 μm thickobtained in Comparative Example 2 was found to be 100% after 1 day,which indicates complete dissolution. It remained completely dissolvedafter 4 days and after 7 days.

Thus, it was found that the solids of hyaluronic acid obtained inComparative Examples dissolved in water quite quickly (Tests Nos.5 to6), whereas the hyaluronic acid gels obtained according to the presentinvention dissolved very slowly (for example, Tests Nos. 1 to 4).

These results suggest that the hyaluronic acid gel obtained according tothe present invention has a long in vivo residence time.

TABLE 1 Degree of dissolution (upper column %) and shape (lower column)of hyaluronic acid gel Test After 4 After 7 After No After 1 day daysdays 14 days Remarks 1 3 5 6 10 Example 1 Spongy Spongy Spongy Spongy 22 4 6 15 Example 2 Spongy Spongy Spongy Spongy 3 9 14  28  38 Example 3Spongy Spongy Spongy Spongy 4 3 5 7 11 Example 4 Spongy Spongy SpongySpongy 5 100  The The The (Completely same same dissolved) condi- condi-condi- Comparative tion as tion as tion as Example 2 after 1 after 1after 1 day day day 6 100  The The The (Completely same same dissolved)condi- condi- condi- Comparative tion as tion as tion as Example 3 after1 after 1 after 1 day day day

COMPARATIVE EXAMPLE 4

A powder of sodium hyaluronate with a molecular weight of 2×10⁶ Da wassubjected to a solubility test for hyaluronic acid gels.

COMPARATIVE EXAMPLE 5

A powder of sodium hyaluronate with a molecular weight of 2×10⁶ Da waspress-molded into discoidal pellets. The pellets were subjected to asolubility test for hyaluronic acid gels.

EXAMPLE 7 Solubility Test for Hyaluronic Acid Gels

A phosphate buffer was added to physiological saline to a concentrationof 50 mM to give a phosphate buffer-physiological saline at pH 7.0. Thespongy hyaluronic acid gels obtained in the preceding Examples werewashed with distilled water and drained on filter paper. The hyaluronicacid gels were immersed in 50 ml of the phosphate buffer-physiologicalsaline based on 20 mg of dry hyaluronic acid in the gels.

The solids of hyaluronic acid obtained in Comparative Examples wereimmersed in 50 ml of the phosphate buffer-physiological saline based on20 mg of dry weight.

The degree of dissolution of hyaluronic acid in the phosphatebuffer-physiological saline at 37° C. with stirring was obtained fromthe concentration of hyaluronic acid in the phosphatebuffer-physiological saline.

Namely, the solubility of a hyaluronic acid gel in a neutral aqueoussolution at 37° C. is defined according to this test.

As described above, a solubility test was actually carried out on thehyaluronic acid gels obtained in Examples 1 to 4 and the solids ofhyaluronic acid obtained in Comparative Examples 2 to 5. The resultstabulated in Table 2.

TABLE 2 Degree of dissolution of hyaluronic acid gel (%) Test After 6After 12 After 24 No. hours hours hours Remarks 7 12 14 16 Example 1 812 16 19 Example 2 9 13 22 23 Example 3 10 12 15 18 Example 4 11 100 Thesame The same Comparative (Completely condition as condition as Example2 dissolved) after 6 hours after 6 hours 12 100 The same The sameComparative (Completely condition as condition as Example 3 dissolved)after 6 hours after 6 hours 13 100 The same The same Comparative(Completely condition as condition as Example 4 dissolved) after 6 hoursafter 6 hours 14 100 The same The same Comparative (Completely conditionas condition as Example 5 dissolved) after 6 hours after 6 hours

For example, in Test No.7, the degree of dissolution of the hyaluronicacid gel obtained in Example 1 was found to be 14% after 12 hours and16% after 24 hours. Namely, 84% of the hyaluronic acid remained evenafter 24 hours. In contrast, in Test No.11, the degree of dissolution ofthe cast film of about 100 μm thick obtained in Comparative Example 2was found to be 100% after 6 hours, which indicates completedissolution.

Thus, it was found that the solids of hyaluronic acid obtained inComparative Examples dissolved in water quite quickly (Tests Nos.11 to14), whereas the hyaluronic acid gels obtained in accordance with thepresent invention dissolved quite slowly (for example, Tests Nos. 7 to10). These results suggest that the hyaluronic acid gel obtainedaccording to the present invention has a long in vivo residence time.

EXAMPLE 8 Solubilization Test for Hyaluronic Acid Gels

The pH of distilled water was adjusted to 1.5 with hydrochloric acid.The spongy hyaluronic acid gel obtained in Example 1 was washed withdistilled water, then washed in the phosphate buffer-physiologicalsaline mentioned in Example 6 and washed with distilled water. Thewashed hyaluronic acid gel was freeze-dried. The resulting hyaluronicacid gel was immersed in 15 ml of the aqueous solution at pH 1.5, basedon 15 mg of dry hyaluronic acid. The solution was left in an oven set at60° C. 0.5 ml samples were withdrawn after 2 hours, after 6 hours andafter 12 hours. After 6 hours, the hyaluronic acid gel had disappearedalmost completely and was not visually recognizable.

COMPARATIVE EXAMPLE 6

Sodium hyaluronate with a molecular weight of 2×10⁶ Da was dissolved indistilled water to give a 0.1 wt % hyaluronic acid aqueous solution. ThepH of the aqueous solution was adjusted to 1.5 with 1N hydrochloricacid. A 15 ml portion of the acidic hyaluronic acid aqueous solution wasleft in an oven at 60° C. for 4 hours for acid hydrolysis of the linearhyaluronic acid.

EXAMPLE 9 Measurement of Molecular Weight and Branching Degree ofSolubilized Hyaluronic Acid

For GPC-MALLS measurement, the solubilized hyaluronic acid obtained inExample 8 and the acid hydrolysate of linear hyaluronic acid obtained inComparative Example 6 were diluted by a factor of 2 with the GPC eluentto 0.05 wt % and filtered through a membrane filter of 0.2 μm, and 0.1ml portions of them were injected.

The measurement was carried out by using a GPC column SB806HQ (ShowaDenko K.K.), a differential refractometer 830-RI (JASCO Corporation) asa detector, a MALLS DAWNDSP-F (Wyatt), 0.2M aqueous sodium nitrate asthe eluent at a temperature of 40° C. at a flow rate of 0.3 ml/min atintervals of 1 datum/2 sec. For the measurement of the intensity ofscattering, eight detectors with scattering angles from 21.7° to 90°were used. For data processing, software ASTRA Version 4.10 (Wyatt) wasused.

As described above, the solubilized hyaluronic acid obtained in Example8 and the acid hydrolysate of linear hyaluronic acid obtained inComparative Example 6 were examined. The results are shown in Table 3.

TABLE 3 Weight- Molecular Degree of Reaction average weight solubili-Test Time molecular distribu- zation No. (hour) weight tion Mw/Mn (%)Remarks 15 2 36.8 × 10⁴ 1.8 28 Example 8 16 6 37.8 × 10⁴ 2.4 86 Example8 17 12 10.7 × 10⁴ 1.8 97 Example 8 Compar- ative 18 4 24.7 × 10⁴ 1.6 —Example 6

For example, in Test No.15, it was found that the hyaluronic acid gelobtained in Example 8 was solubilized to a low degree when withdrawnafter 2 hours of reaction. In Test No.17, the sample withdrawn after 12hours of reaction showed such a low molecular weight that the branchingdegree was difficult to measure. In Test No.16, the hyaluronic acid gelwas solubilized to a high degree when withdrawn after 6 hours ofreaction, and the large molecular weight distribution of 2.4 reflectsthe presence of branched hyaluronic acid.

The GPC chromatograms of the solubilized hyaluronic acid obtained inExample 8 after 6 hours of reaction and the acid hydrolysate of linearhyaluronic acid obtained in Comparative Example 6, and the results ofcalculation of their branching degrees obtained in Test No. 16 and TestNo.18, respectively, were shown in FIG. 1 and FIG. 2.

As is evident from FIG. 1, the GPC chromatogram 1 for Example 8 had ashoulder at a higher molecular weight range than the GPC chromatogram 2for Comparative Example 6. From comparison of the molecular weights offractions at the same elution volumes, it was found that the molecularweight for Example 8 was clearly higher than that for ComparativeExample 6 within the elution volume range of at most 8.6 ml, whichcorresponds to the molecular weight range of about 200,000 or larger.

The fractions for Example 8 showed higher molecular weights than thefractions for Comparative Example 6 at the same elution volumes, becauseof the presence of branched hyaluronic acid.

FIG. 2 shows the relation of the branching degree and the molecularweight for Example 8 calculated on the basis of the linear hyaluronicacid of Comparative Example 6. The branching degree was calculated fromthe molecular weights of fractions for Example 8 and Comparative Example6 at the same elution volumes by using equations (2) and (3).

FIG. 2 shows a sharp rise in the branching degree from 0.5 within themolecular weight range of 200,000 or larger for Example 8, whichindicates that the hyaluronic acid gel obtained according to the presentinvention contains a crosslinked structure stable under acceleratingconditions for acid hydrolysis of hyaluronic acid.

EXAMPLE 10 Immersion Test for Hyaluronic Acid Gels in an AlkalineBuffered Aqueous Solution

The spongy hyaluronic acid gel obtained in Example 1 was washed withdistilled water, then washed in the phosphate buffer-physiologicalsaline mentioned in Example 6, and washed with distilled water. Thewashed hyaluronic acid gel immersed and left in 50 ml, based on 150 mgof dry hyaluronic acid in the gel, of 25 mM disodiumhydrogenphosphate-sodium hydroxide buffer at pH 11 at 25° C., and as aresult, the gel dissolved quickly and completely dissolved in 1 hour.Similarly, when the gel was immersed in 25 mM sodiumhydrogencarbonate-sodium hydroxide buffer at pH 10, it lost shape in 7hours and completely dissolved in 18 hours.

It was found that the hyaluronic acid gel obtained according to thepresent invention had a feature that it is hardly soluble in a neutralaqueous solution but dissolves quickly in an alkaline aqueous solution.

EXAMPLE 11 Measurement of the Swelling Ratio of a Hyaluronic Acid Gel

The spongy hyaluronic acid gel obtained in Example 1 was washed withdistilled water, then washed in the phosphate buffer-physiologicalsaline mentioned in Example 6 and washed with distilled water. Then, thewashed hyaluronic acid gel was freeze-dried.

100 mg, on a dry basis, of the hyaluronic acid gel was immersed in 200ml of distilled water and left to stand at 25° C. for 24 hours. Theswollen hyaluronic acid gel was withdrawn, drained on filter paper andweighed. The swelling ratio was 117.

The hyaluronic acid gel obtained according to the present invention wasfound to have a measurably stable swelling ratio.

EXAMPLE 12 Test on Cytotoxicity of a Hyaluronic Acid Gel

The cytotoxicity of the hyaluronic acid gel obtained according to thepresent invention was evaluated by observing the proliferation behaviorof a normal human skin-derived fibroblast culture in the presence of thehyaluronic acid gel obtained according to the present invention withoutcontact between them. The spongy hyaluronic acid gel obtained in Example1 was freeze-dried in the same manner as in Example 8. The freeze-driedgel was mechanically pulverized, and 20 mg of the pulverized gel wasloaded on a cell culture insert (pore size: 3 μm, Falcon) and immersedin the cell culture. For a control experiment, incubation was carriedout in the absence of the hyaluronic acid.

Incubation conditions

Plate: 12-well plate for cell culture

Medium: PDMEM medium+10% fetal bovine serum, 2 ml/well

Temperature: 37.5° C. (under 5% CO₂)

Cell number: 1×10⁴ cells/well

After 2, 5 and 8 days of incubation, the cell culture was examined onthe cell density under an inverted microscope. As a result, it was foundthat the cell culture had grown in the presence of the hyaluronic acidgel as satisfactorily as that in the control experiment, and thereby itwas ascertained that the hyaluronic acid gel obtained according to thepresent invention had no cytotoxicity.

EXAMPLE 13

Sodium hyaluronate with a molecular weight of 2×10⁶ Da was dissolved indistilled water to give a 1 wt % hyaluronic acid aqueous solution. ThepH of the aqueous solution was adjusted to 1.5 with 1N hydrochloric acidto give an acidic hyaluronic acid aqueous solution. A 25 ml portion ofthe acidic hyaluronic acid aqueous solution was put in a plastic Petridish and placed in a refrigerator set at −20° C. 22 hours of freezingand 2 hours of thawing at 25° C. were repeated twice to give a spongyhyaluronic acid gel. Then, the gel was immersed for neutralization at 5°C. for 24 hours in 100 ml of a phosphate buffer-physiological saline atpH 7 prepared by adding a phosphate buffer to physiological saline to aconcentration of 50 mM, and washed thoroughly with distilled water. Thegel was pressed between two plates and freeze-dried to give an adhesionpreventive sheet made of a hyaluronic acid gel.

EXAMPLE 14

The procedure in Example 13 was followed except that sodium hyaluronatewith a molecular weight of 6×10⁵ Da was used to give an adhesionpreventive sheet made of a hyaluronic acid gel.

EXAMPLE 15

A 25 ml portion of the acidic aqueous solution of hyaluronic acid with amolecular weight of 2×10⁶ Da at pH 1.5 prepared in Example 13 was put ina plastic Petri dish and placed in a refrigerator set at −20° C. 22hours of freezing and 2 hours of thawing at 25° C. were repeated twiceto give a spongy hyaluronic acid gel. Then, the gel was immersed in aphosphate buffer-physiological saline, washed and pressed between twoplates in the same manners as in Example 13. Then, the gel was air-driedat 30° C. for 3 hours to give an adhesion preventive film made of ahyaluronic acid gel.

EXAMPLE 16

50 mg of the hyaluronic acid sheet obtained in Example 13 wasaseptically crushed in 10 ml of physiological saline in amicrohomogenizer (POLYTORON, KINIMATICA AG) to give a flaky adhesionpreventive made of a hyaluronic acid gel.

EXAMPLE 17

A 15 ml portion of the acidic aqueous solution of hyaluronic acid with amolecular weight of 2×10⁶ Da at pH 1.5 prepared in Example 13 was put ina 30 ml vessel and placed in a refrigerator set at −20° C. 22 hours offreezing and 2 hours of thawing at 25° C. were repeated twice to give aspongy hyaluronic acid gel. Then, the gel was immersed forneutralization at 5° C. for 24 hours in 100 ml of a phosphatebuffer-physiological saline at pH 7 prepared by adding a phosphatebuffer to physiological saline to a concentration of 50 mM, and washedthoroughly with distilled water. The gel was directly freeze-dried togive an adhesion preventive sponge made of a hyaluronic acid gel.

EXAMPLE 18

A 15 ml portion of the acidic aqueous solution of hyaluronic acid with amolecular weight of 2×10⁶ Da at pH 1.5 prepared in Example 13 was put ina 30 ml vessel and placed in a refrigerator set at −20° C. 22 hours offreezing and 2 hours of thawing at 25° C. were repeated twice to give aspongy hyaluronic acid gel. The gel was immersed in a phosphatebuffer-physiological saline and washed in the same manners as in Example13, then drained by centrifugation and freeze-dried in the compactedstate to give a biomedical mass made of a hyaluronic acid gel.

EXAMPLE 19

Sodium hyaluronate with a molecular weight of 2×10⁶ Da was dissolved indistilled water at a concentration of 0.05%, and the pH was adjusted to1.5 with 1N hydrochloric acid. A 100 ml portion of the acidic hyaluronicaid aqueous solution was put in a 200 ml vessel and placed in arefrigerator set as −20° C.

22 hours of freezing and 2 hours of thawing at 25° C. were repeatedtwice to give a fibrous hyaluronic acid gel. The gel was collected byfiltration, then immersed in a phosphate buffer-physiological saline,washed and freeze-dried in the same manners as in Example 13 to give afibrous biomedical material made of a hyaluronic acid gel.

EXAMPLE 20

A 5 ml portion of the acidic solution of hyaluronic acid with amolecular weight of 2×10⁶ Da at pH 1.5 prepared in Example 13 was pouredinto a tubular mold and placed in a refrigerator set as −20° C. 22 hoursof freezing and 2 hours of thawing at 25° C. were repeated twice to givea tubular hyaluronic acid gel. The gel was immersed in a phosphatebuffer-physiological saline, washed and freeze-dried in the same mannersas in Example 13 to give a biomedical tube made of a hyaluronic acidgel.

EXAMPLE 21

A 25 ml portion of the acidic solution of hyaluronic acid with amolecular weight of 2×10⁶ Da at pH 1.5 prepared in Example 13 was put ina plastic Petri dish and placed in a refrigerator set as −20° C. 22hours of freezing and 2 hours of thawing at 25° C. were repeated twiceto give a spongy hyaluronic acid gel. The gel was immersed in aphosphate buffer-physiological saline and washed in the same manners asin Example 13. After mild drainage, the gel was impregnated with 5 ml ofa 1 wt % hyaluronic acid aqueous solution, pressed between two platesand freeze-dried to give an adhesion preventive sheet of a hyaluronicacid gel coupled with hyaluronic acid.

COMPARATIVE EXAMPLE 7

The hyaluronic acid aqueous solution prepared in Example 13 was adjustedto pH 7.0 with 1N sodium hydroxide, and a 25 ml portion of the solutionwas frozen at −20° C. and freeze-dried in a plastic Petri dish to give ahyaluronic acid sheet.

COMPARATIVE EXAMPLE 8

The hyaluronic acid aqueous solution prepared in Example 13 was adjustedto pH 7.0 with 1N sodium hydroxide, and a 25 ml portion of the solutionwas air-dried at 60° C. in a plastic Petri dish to give a hyaluronicacid sheet.

COMPARATIVE EXAMPLE 9

The hyaluronic acid aqueous solution prepared in Example 13 was adjustedto pH 7.0 with 1N sodium hydroxide, and a 25 ml portion of the solutionwas frozen at 20° C. and freeze-dried in a beaker to give a hyaluronicacid sponge.

COMPARATIVE EXAMPLE 10

In a solution of 1.1 g of disodium hydrogenphosphate hydrate in 30 g ofwater adjusted to pH 10 with 2% sodium hydroxide, 0.6 g of sodiumhyaluronate with a molecular weight of 6×10⁵ Da was dissolved. Then,0.05 g of cyanuric chloride in 1.5 ml of dioxane was added to theabove-mentioned hyaluronic acid solution, and the reaction was carriedout at room temperature for 3 hours. Then, the reaction solution was putinto a dialysis membrane, dialyzed against water for 1 day, poured ontoa framed glass plate and dried to give a film.

EXAMPLE 22 Solubility Test on Adhesion Preventives Biomedical MaterialsMade of Hyaluronic Acid Gels

A 50 mM phosphate buffer-physiological saline at pH 7 was prepared byadding a phosphate buffer to physiological saline. Adhesion preventivesand biomedical materials made of hyaluronic acid gels containing 150 mgof hyaluronic acid on a dry basis were gently shaken in 50 ml of thephosphate buffer-physiological saline. The solubilities of the adhesionpreventives and biomedical materials made of hyaluronic acid gels in thephosphate buffer-physiological saline at 25° C. were evaluated fromtheir shapes.

As described above, the solubility test was actually carried out on theadhesion preventives and biomedical materials made of hyaluronic acidgels obtained in Examples 13 to 21 and comparative Examples 7 to 9. Theresults are shown in Table 4.

TABLE 4 Shape of adhesion preventives and biomedical material made ofhyaluronic acid After 4 After 7 Test No. After 1 day days days Remarks19 Not changed Not changed Partly Example 13 dissolved 20 Not changedPartly Partly Example 14 dissolved dissolved 21 Not changed Not changedPartly Example 15 dissolved 22 Not changed Partly Partly Example 16dissolved dissolved 23 Not changed Not changed Not Example 17 changed 24Not changed Not changed Not Example 18 changed 25 Not changed Notchanged Partly Example 19 dissolved 26 Not changed Not changed PartlyExample 20 dissolved 27 Not changed Partly Partly Example 21 dissolveddissolved 28 Completely The same as The same Comparative dissolved thestate as the Example 7 after 1 day state after 1 day 29 Completely Thesame as The same Comparative dissolved the state as the Example 8 after1 day state after 1 day 30 Completely The same as The same Comparativedissolved the state as the Example 9 after 1 day state after 1 day

As shown in Table 4, the adhesion preventives and biomedical materialsmade of hyaluronic acid gels obtained in Examples (Tests Nos.19 to 27)did not change or partly dissolved in 7 days in the neutral aqueoussolution at 25° C. and were able to keep their shapes for at least 1day, whereas the mere molded sheets and sponges of hyaluronic acidobtained in Comparative Examples (Tests Nos.28 to 30) dissolvedcompletely in 1 day.

EXAMPLE 23 Solubility Test on Adhesion Preventives Biomedical MaterialsMade of Hyaluronic Acid Gels

A 50 mM phosphate buffer-physiological saline at pH 7 was prepared byadding a phosphate buffer to physiological saline. Adhesion preventivesand biomedical materials made of hyaluronic acid gels containing 150 mgof hyaluronic acid on a dry basis were gently shaken in 50 ml of thephosphate buffer-physiological saline. The proportions of the hyaluronicacid dissolved in the phosphate buffer-physiological saline at 37° C.were calculated from the hyaluronic acid concentrations of the phosphatebuffer-physiological saline.

Measurement of Hyaluronic Acid Concentration

The concentration of hyaluronic acid in the phosphatebuffer-physiological saline was obtained from the area of a GPC peak byusing a differential refractometer as a detector.

As described above, the solubility test was actually carried out on theadhesion preventives and biomedical materials made of hyaluronic acidgels obtained in Examples 13 to 21 and Comparative Examples 7 to 9. Theresults are shown in Table 5.

TABLE 5 Degree of dissolution of adhesion preventive and biomedicalmaterial made of hyaluronic acid (%) Test After 12 After 1 After 4 After7 No hours day days days Remarks 31 6 15 24 29 Example 13 32 15 21 38 55Example 14 33 6 18 26 35 Example 15 34 8 21 29 40 Example 16 35 8 16 2228 Example 17 36 6 15 20 26 Example 18 37 8 15 26 32 Example 19 38 8 1424 30 Example 20 39 12 21 30 36 Example 21 40 100 100 100 100Comparative Example 7 41 100 100 100 100 Comparative Example 8 42 96 100100 100 Comparative Example 9

As shown in Table 5, the adhesion preventives and biomedical materialsmade of hyaluronic acid gels obtained in Examples (Tests Nos.31 to 39)dissolved to degrees of dissolution of 26 to 55% in 7 days in theneutral aqueous solution at 25° C. and were hardly soluble, whereas themere molded sheets and sponges of hyaluronic acid obtained inComparative Examples (Tests Nos.40 to 42) dissolved to degrees of 96 to100% in 12 hours.

EXAMPLE 24 Biocompatibility Test and In Vivo Persistency Test onAdhesion Preventive Hyaluronic Acid Gels

For the following test, the adhesion preventive hyaluronic acid sheetsobtained in Examples 13 and 14 cut into 1 cm×1 cm squares, and ascontrols, the hyaluronic acid sheet obtained in Comparative Example 7and the cyanuric chloride-crosslinked hyaluronic acid obtained inComparative Example 10 were cut into 1 cm×1 cm squares.

Five of twenty 12-week-old female DDY mice (average body weight 33 g)were used for implantation of the hyaluronic acid gel (molecular weight2×10⁶ Da), five for implantation of the hyaluronic acid gel (molecularweight 6×10⁵ Da), and in comparative tests, five were used for thefreeze-dried hyaluronic acid, and the remaining five for the cyanuricchloride-crosslinked hyaluronic acid.

In the implantation, mice were cut about 1.5 cm long along theventrimeson under a nembutal anesthetic and then sutured with variouskinds of hyaluronic acid placed on the appendices.

3, 5, 7, 9 and 14 days after the implantation, one of the mice implantedwith each kind of hyaluronic acid gel and freeze dried hyaluronic acidwas killed by cervical dislocation and cut on the abdomen. Then, thestate of the implantation site was observed. Then, the inside of theintraperitoneal cavity was washed with physiological saline to recoverthe remaining hyaluronic acid, inclusive of the residue of thehyaluronic acid sheet.

The recovered washings were mixed with the equal amount of 0.02N sodiumhydroxide, then left to stand for 1 hour and neutralized withhydrochloric acid. They were subsequently centrifuged and filteredthrough a filter (pore size 0.45 μm) to give. The resulting samples wereanalyzed by GPC to determine hyaluronic acid in the samples. Therecoveries of hyaluronic acid based on the hyaluronic acid in theimplanted sheets were calculated and shown in Table 6 together with theresults of the observation of the states of the implantation sites.

TABLE 6 Recovery of Days of hyaluro- State Test Implanted implan- nicacid State of of No. sheet tation (%) sheet tissue 43 Hyaluronic 3 83Original ◯ acid of shape Example 13 6 5 63 Original ◯ (M.W. 2 × 10⁶)shape 7 28 Fragmented ◯ 9 15 Small ◯ residue 14 0 Undetect- ◯ able 44Hyaluronic 3 59 Original ◯ acid of shape Example 14 5 13 Small ◯ (M.W. 6× 10⁵) residue ◯ 7 2 Small ◯ residue 9 0 Undetect- ◯ able 14 0 Undetect-◯ able 45 Freeze dried 3 0 Undetect- ◯ hyaluronic able acid of 5 0Undetect- ◯ Comparative able Example 7 7 0 Undetect- ◯ able 9 0Undetect- ◯ able 14 0 Undetect- ◯ able 46 Cyanuric 3 73 Original Δchloride- shape crosslinked 5 46 Original Δ hyaluronic shape acid of 710 Small Δ Comparative residue Example 10 9 0 Undetect- Δ able 14 0Undetect- Δ able O: normal A: slight inflammation

Although all the mice grew normally, slight inflammation was observed onthe tissues implanted with cyanuric chloride-crosslinked hyaluronic acidobtained in Comparative Example 10, whereas the tissues implanted withthe hyaluronic acid gels and the freeze dried hyaluronic acid werenormal.

EXAMPLE 25 Test on Adhesion Preventive Effect of Adhesion PreventiveHyaluronic Acid Gels Using a Mouse Uterine Model

For the following test, the adhesion preventive hyaluronic acid gelobtained in Example 13 was cut into 1 cm×2 cm rectangles, and similarlythe adhesion preventive hyaluronic acid gel obtained in Example 13impregnated with the hyaluronic acid solution prepared in Example 13 andas controls, the hyaluronic acid sheet obtained in Comparative Example7, the hyaluronic acid solution prepared in Example 13 and the cyanuricchloride-crosslinked hyaluronic acid obtained in Comparative Example 10were cut into 1 cm×2 cm rectangles.

7-week-old female ICR mice (body weight 25 to 30 g) were anesthetized byintraperitoneal pentobarbital injection and cut along the ventrimeson.Then, an abrasion of about 10 cm long was made on the uterine horn ofeach mouse by application of iodine. Ten mice were allotted to eachtreatment group. The above-mentioned 1 cm×2 cm rectangular sheets of ahyaluronic acid gel, hyaluronic acid or cyanuric chloride-crosslinkedhyaluronic acid or nothing, for control test, were wrapped around theabrasions. In the case of the hyaluronic acid solution, a 1 ml portionof the hyaluronic acid solution prepared in Example 13 was applied toeach abrasion. In the case of the combined use of the hyaluronic acidgel and the hyaluronic acid solution, the hyaluronic acid gel waswrapped around the abrasions first, and the hyaluronic acid solution wasadded to the intraperitoneal cavity. In any case, 5-0 Dexon was used forclosure.

10 days later, each group of mice, which were not treated or treatedwith the hyaluronic acid gel, the combination of the hyaluronic acid geland the hyaluronic acid solution, the hyaluronic acid sheets, thehyaluronic acid solution or the cyanuric chloride-crosslinked hyaluronicacid, were sacrificed by cervical dislocation. Then ventrotomy wasperformed again, and inspection for adhesions was carried out. In thejudgement of formation of an adhesion, very slight membranous adhesionswere excluded, and only fibrous and thick adhesions strong enough not topeel off even if pulled with tweezers were counted in. The results areshown in Table 7.

TABLE 7 Adhesion Test formation No. Group ratio Remarks 47 No treatment9/10 Comparative Example 48 Hyaluronic acid gel of 1/10 Example Example13 49 Hyaluronic acid gel of 0/10 Example Example 13 and hyaluronic acidsolution prepared in Example 13 50 Hyaluronic acid sheet 5/10Comparative of Comparative Example Example 7 51 Hyaluronic acid 6/10Comparative solution prepared in Example Example 13 52 Cyanuricchloride- 3/10 Comparative crosslinked hyaluronic Example acid ofComparative Example 10

As shown in FIG. 7, formation of adhesions was recognized in nine of theten non-treated mice, in five of the ten treated with the merehyaluronic acid sheets, in six of the ten treated with the hyaluronicacid solution and three of the ten treated with the cyanuricchloride-crosslinked hyaluronic acid, whereas the adhesion preventivehyaluronic acid gel prepared in Example 13 and the combination of theadhesion preventive hyaluronic acid gel prepared in Example 13 and thehyaluronic acid solution prepared in Example 13 developed adhesions inone and none, respectively, of the mice treated. Thus, it was suggestedthat the adhesion preventive hyaluronic acid gel prepared in Example 13and the combination of the adhesion preventive hyaluronic acid gelprepared in Example 13 and the hyaluronic acid solution prepared inExample 13 have strong adhesion preventive effect.

EXAMPLE 26 Adhesion Preventive Test of Adhesion Preventive HyaluronicAcid Gels on a Rat Cecal Model

For the following test, the adhesion preventive hyaluronic acid gelobtained in Example 13, and as controls, the hyaluronic acid sheetobtained in Comparative Example 7 and the cyanuric chloride-crosslinkedhyaluronic acid were cut into 2 cm×2 cm squares.

10-week-old male Wister rats (body weight about 250 g) were anesthetizedwith ketamine (60 mg/l kg body weight) and xylazine (10 mg/l kg bodyweight) intraperitoneally and cut along the ventrimeson. The ceca wereabraded over about 10 cm×10 cm with a gauge (about 20 times) to developabrasions with blood spots. In each group of five rats, the abrasionswere covered with nothing (control), or the above-mentioned 2 cm×2 cmrectangular sheets of the hyaluronic acid gel, hyaluronic acid orcyanuric chloride-crosslinked hyaluronic acid, and closure was performedby using 3-0 Dexon.

14 days later, the five rats in each group treated with nothing, thehyaluronic acid gel, the hyaluronic acid sheet or the cyanuricchloride-crosslinked hyaluronic acid were sacrificed. Then ventrotomywas performed again, and inspection for adhesions was carried out. Inthe judgement of formation of an adhesion, very slight membranousadhesions were excluded, and only fibrous and thick adhesions strongenough not to peel off even if pulled with tweezers were counted in. Theresults are shown in Table 8.

TABLE 8 Adhesion Test formation No. Group ratio Remarks 53 No treatment4/5 Comparative Example 54 Hyaluronic acid gel of 1/5 Example Example 1355 Hyaluronic acid sheet of 3/5 Comparative Comparative Example 7Example 56 Cyanuric chloride- 2/5 Comparative crosslinked hyaluronicExample acid of Comparative Example 10

As shown in FIG. 8, formation of adhesions was recognized in four of thefive non-treated rats, in three of the five treated with the merehyaluronic acid sheets, and in two of the ten treated with the cyanuricchloride-crosslinked hyaluronic acid, whereas the adhesion preventivehyaluronic acid gel prepared in Example 13 developed adhesions in one ofthe rats treated.

As described above, according to the present invention, it is possibleto obtain a hyaluronic acid gel hardly soluble in water without usingany chemical crosslinkers or chemical modifiers. The adverse effects onbiocompatibility attributable to chemical crosslinkers or chemicalmodifiers are avoided, and the hyaluronic acid gel is useful in thefield of biocompatible materials by virtue of its long in vivo residencetime. In particular, the hyaluronic acid gel which is hardly soluble inwater can provide an excellent adhesion preventive which (1) has idealin vivo persistency as an adhesion preventive, (2) prevents effectivelypostoperative adhesion by virtue of the drastically improved residencetime on wounds, and (3) is so safe as to thoroughly solve the problemsof conventional chemically modified hyaluronic acid with toxicity andbiocompatibility.

What is claimed is:
 1. A gel comprising hyaluronic acid, or its alkalimetal salt, said gel prepared by at least once freezing and subsequentlythawing a hyaluronic acid aqueous solution at a pH of 3.5 or below,which has not been subjected to chemical crosslinking by a chemicalcrosslinking compound other than hyaluronic acid or chemicalmodification by a chemical modifying compound other than hyaluronicacid, and which is not in the form of a complex with a cationic polymerand (1) which dissolves in a neutral aqueous solution at 25° C. in oneday to a degree of dissolution of at most 50%, or (2) which dissolves ina neutral aqueous solution at 37° C. in 12 hours to a degree ofdissolution of at most 50%, or both (1) and (2).
 2. The hyaluronic acidgel according to claim 1, which keeps its shape for at least one day ina neutral aqueous solution at 25° C.
 3. The hyaluronic acid gelaccording to claim 1, which is obtained by acid hydrolysis of hyaluronicacid under conditions wherein said hyaluronic acid gel dissolves toyield solubilized hyaluronic acid having a branched structure andcontaining a molecular weight fraction with a branching degree of atleast 0.5.
 4. The hyaluronic acid gel according to claim 3, wherein saidconditions comprise a pH of an aqueous solution to be 1.5 and atemperature of 60° C.
 5. A biomedical material containing a gel made ofhyaluronic acid or its alkali metal salt, said gel prepared by at leastonce freezing and subsequently thawing a hyaluronic acid aqueoussolution at a pH of 3.5 or below, wherein said gel has not beensubjected to chemical crosslinking by a chemical crosslinking compoundother than hyaluronic acid or chemical modification by a chemicalmodifying compound other than hyaluronic acid, and which is not in theform of a complex with a cationic polymer which satisfies the followingrequirements (a) and (b): (a) the hyaluronic acid gel dissolves in aneutral aqueous solution at 25° C. in one day to a degree of dissolutionof at most 50%, and (b) the gel is obtained by acid hydrolysis ofhyaluronic acid under conditions such that said hyaluronic acid geldissolves to yield solubilized hyaluronic acid having a branchedstructure and containing a molecular weight fraction with a branchingdegree of at least 0.5.
 6. The biomedical material according to claim 5,wherein the gel made of hyaluronic acid is in the form of a sheet, afilm, a flake, a sponge, a mass, a fiber or a tube.
 7. The biomedicalmaterial according to claim 5, which is an adhesion preventive.
 8. Abiomedical material containing a hyaluronic acid, or its alkali metalsalt, gel and an un-gelled hyaluronic acid, said gel prepared by atleast once freezing and subsequently thawing a hyaluronic acid aqueoussolution at a pH of 3.5 or below and wherein said gel has not beensubjected to chemical cross-linking by a chemical cross-linking compoundother than hyaluronic acid or chemical modification by a chemicalmodifying compound other than hyaluronic acid, and said gel is not inthe form of a complex with a cationic polymer, wherein the hyaluronicacid gel dissolves in a neutral aqueous solution at most 50%, and thehyaluronic acid gel is obtained by acid hydrolysis of hyaluronic acidunder conditions such that said hyaluronic acid gel dissolves to yieldsolubilized hyaluronic acid having a branched structure and containing amolecular weight fraction with a branching degree of at least 0.5.
 9. Abiomedical material according to claim 8, wherein said conditionscomprise a pH of an aqueous solution to be 1.5 and a temperature of 60°C.
 10. A biomedical material containing, in the form of a sheet, a film,a flake, a sponge, a mass, a fiber or a tube, (1) hyaluronic acid gelmade of hyaluronic acid, or its alkali metal salt, said gel prepared byat least once freezing and subsequently thawing a hyaluronic acidaqueous solution at a pH of 3.5 or below, and wherein said gel has notbeen subjected to chemical crosslinking or chemical modification by acompound other than hyaluronic acid, and which is not in the form of acomplex with a cationic polymer and (2) an un-gelled hyaluronic acid.